Reversible methylation of cytosines is a major epigenetic modification in multicellular organisms and is found in many human diseases including cancer. Cancer epigenomes are found to be globally hypomethylated with promoter-specific hypermethylations. Furthermore, cytosine methylation results in transcriptional repression, which, in the case of tumour suppressor genes, apoptotic genes, DNA repair genes and factors controlling cell cycle check points leads to tumour progression.
Prostate cancer (PC) is the third most common cause of male cancer deaths in developed countries. Diagnosed at an early stage PC is a curable disease. Therapies reach from watchful waiting to radical prostatectomy, hormone or radiation therapy. Nevertheless, because of its yet mostly unpredictable outcome patients are often treated without clear benefit.
Prostate specific antigen (PSA) is used as a biomarker to screen men for potential tumour development. However, low specificity and sensitivity leads to wrong diagnoses. In particular, elevated PSA can also result from an inflammation or precedent transrectal ultrasound, i.e. disclosure within the state of the art lacks an unequivocal diagnosis of PC.
It is therefore clear that there has been and remains today a long standing need for an accurate and reliable test to diagnose PC.
Recent years have brought a marked extension of our understanding of the somatic basis of prostate cancer. With one to three mutations per megabase the mutation frequency is similar to that observed in acute myeloid leukemia and breast cancer and lies within the lower range of cancer. Based on the frequency and the fact that primarily a diverse array of genes is affected the main genomic alterations appear to be genomic rearrangements and changes in the epigenetic structure of the DNA.
Michael Weber et al. (Nature Genetics, Vol. 37, No. 8, Aug. 1, 2005, pages 853-862) disclose DNA methylation markers for colorectal cancer. For the analyses colorectal cancer cell lines and tissues, and a prostate cancer cell line were used, but no primary prostate cancer tissues. In addition, experiments are based on arrays which narrow down the possibility to detect new DMRs.
Ilana Keshet et al (Nature Genetics, Vol. 38, No. 2, Feb. 1, 2006, pages 149-153) disclose methylation of GSTP1 and CDKN2A in Caco2 and PC3 cell lines (has been shown before) by PCR analyses. Furthermore, they compare methylation information obtained by MeDIP array (10.000 promoter elements) analyses of PC3, Caco2, normal lymphoblasts, normal colon tissue and six colon tumors. Again, no primary prostate cancer tissues have been used, and experiments are restricted to array regions.
Tokumaru (Clinical Cancer Research, Vol. 10, No. 16, Aug. 15, 2004, pages 5518-5522) disclose the usability of combinations of the methylation values of four genes obtained by qPCR to help in tumour classification of needle biopsies: Combinations of RARRES1, APC, RARB2, and GSTP1 methylation were used to classify 72 prostate samples (56 cancer, only 16 normal) with 100% specificity and 97% sensitivity. RARB2 as well as GSTP1 are listed in our patent application (SEQ ID NO: 36 and 108).
Hoque Mohammad Obaidul et al (Journal of clinical oncology, American Society of Clinical Onoclogy, Vol. 23, No. 27, Sep. 20, 2005, pages 6569-6575) disclose the usability of the methylation values of CDKN2A, p14ARF, MGMT, and GSTP1 as markers for qMSP based PCa detection in urinary sediment in 52 PCa cases and 91 age matched controls without precedent PCa history with a sensitivity of 87% and a specificity of 100%. Only GSTP1 may overlap with the regions listed below.
Jacinto Filipe V et al (Biotechniques, January 2008, Vol. 44, No. 1) review MeDIP experiments
Ke Xi-Song et al (BMC Genomics, Biomed Central, London, Vol. 11, No, 1, Nov. 25, 2010) performed array profiling (17.000 RefSeq genes −5.5 kb-+2.5 kb) of histone modifications (chromatin IP) and DNA methylation (MeDIP) in EP156T, EPT1 and EPT2 cells and correlated the results to gene expression profiles (Agilent 44 k chip) and disclose histone modifications with correlating gene expression changes during epithelial to mesenchymal transition (EMT). Here only cell lines are used, no DNA methylation PCa markers are named.
Matheys Lesley A et al (Molecular Cancer, Biomed Central, London, GB, Vol. 9, No. 1, October 2010) disclose hypomethylation in BMX and SOX1 in the invasive subpopulations of LNCaP and DU145 cells causing overexpression of said genes and name alterations in the STAT3 pathway as key features of invasiveness. Using MeDIP array analyses they found 869 genes hypermethylated in invasive LNCaP (1015 in invasive DU145) and 44 genes hypomethylated in invasive LNCaP and DU145 cells. They compared subpopulations of prostate cancer cell lines and did not include prostate tissue specimens to validate the significance of their findings in clinical samples.
U.S. Pat. No. 6,812,339 disclose single nucleotide polymorphisms (SNPs) in genes that have been identified as being involved in pathologies associated with human disease. The diseases/pathologies that each gene is known in the art to be associated with is specifically indicated in Table 1 therein. The US patent does not relate to methylation patterns at all and uses a different approach. A nucleic acid disclosed therein shares some similarity with SEQ ID NO. 1 herein.
Aberrant DNA methylation plays an important role in prostate cancer development and seems to be one of the earliest events in tumourigenesis. The most prominent differentially methylated gene in prostate cancer is glutathione S-transferase pi 1 (GSTP1).
Other genes with changes in promoter methylation include multidrug resistance protein 1 (MDR1), O-6-methylguanine-DNA methyltransferase (MGMT), Ras association domain family member 1 (RASSF1), retinoic acid receptor beta (RARB), adenomatous polyposis coli (APC), androgen receptor (AR), cyclin-dependent kinase inhibitor 2A (CDKN2A), E-cadherin (CDH1) and CD44, but show inconsistent methylation levels in different studies.
Even though the relevance of DNA methylations for normal cell homeostasis is undeniable, little is known about the genomic distribution in normal and diseased states. Accordingly, there is a need in the state of the art of studying genome-wide aberrant DNA methylation that can be associated with high confidence to PC and identifying biomarkers for PC diagnosis based on the epigenetic cancer information.